Nobe Moeka, Maruzuru Yuhei, Takeshima Kosuke, Maeda Fumio, Kusano Hideo, Yoshimura Raiki, Nishiyama Takara, Park Hyeongki, Kozaki Yoshitaka, Iwami Shingo, Koyanagi Naoto, Kato Akihisa, Natsume Tohru, Adachi Shungo, Kawaguchi Yasushi
Division of Molecular Virology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.
Department of Infectious Disease Control, International Research Center for Infectious Diseases, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.
mBio. 2025 Jun 11;16(6):e0028025. doi: 10.1128/mbio.00280-25. Epub 2025 May 5.
Although viral protein expression and progeny virus production were independently shown to be highly heterogeneous in individual cells, their direct relationship, analyzed by considering their heterogeneities, has not been investigated to date. To elucidate the direct relationship between viral protein expression and progeny virus production, we constructed a reporter herpes simplex virus 1 (HSV-1) by tagging Venus to the late protein Us11. We then separated the HSV-1-infected cell population into multiple subpopulations according to the fluorescence intensity of Venus-which reflected the expression of L proteins, largely constituting virion structural proteins-and titrated virus yields and performed electron microscopic analysis in each subpopulation. Our results revealed that infectious progeny virus production, as well as nucleocapsid maturation, was triggered only when L protein expression exceeded a specific threshold. This suggested the existence of a rate-limiting step in progeny virus production, with nucleocapsid maturation potentially being one such step.IMPORTANCEEarlier single-cell studies of virus-infected cells have revealed high heterogeneity in the state of viral gene expression and progeny virus yield. Notably, these two aspects have been shown independently, and therefore, the direct relationship between progeny virus production and viral gene expression has been unclear. This study, for the first time, demonstrated the direct and quantitative relationship between viral protein expression and progeny virus production by taking into account their heterogeneities and revealed a threshold for the levels of herpes simplex virus 1 protein expression for progeny virus production, thereby suggesting the existence of a rate-limiting step in progeny virus production.
尽管病毒蛋白表达和子代病毒产生在单个细胞中被独立证明具有高度异质性，但考虑到它们的异质性对其直接关系进行分析，至今尚未有研究。为了阐明病毒蛋白表达与子代病毒产生之间的直接关系，我们通过将金星蛋白（Venus）标记到晚期蛋白Us11上构建了一种报告单纯疱疹病毒1型（HSV-1）。然后，我们根据金星蛋白的荧光强度将感染HSV-1的细胞群体分离为多个亚群，金星蛋白的荧光强度反映了主要构成病毒粒子结构蛋白的L蛋白的表达情况，并对每个亚群的病毒产量进行了滴定，并进行了电子显微镜分析。我们的结果表明，只有当L蛋白表达超过特定阈值时，才会触发有感染性的子代病毒产生以及核衣壳成熟。这表明在子代病毒产生过程中存在一个限速步骤，核衣壳成熟可能就是这样一个步骤。
早期对病毒感染细胞的单细胞研究揭示了病毒基因表达状态和子代病毒产量的高度异质性。值得注意的是，这两个方面是独立显示的，因此，子代病毒产生与病毒基因表达之间的直接关系一直不明确。本研究首次通过考虑它们的异质性，证明了病毒蛋白表达与子代病毒产生之间的直接定量关系，并揭示了单纯疱疹病毒1型蛋白表达水平对于子代病毒产生的阈值，从而表明在子代病毒产生过程中存在一个限速步骤。
